Lixia Yue, Ph.D - US grants

DESCRIPTION (provided by applicant): Cardiac fibrosis is a major biological determinant in a variety of cardiac diseases including hypertrophy, heart failure, severe arrhythmias and sudden cardiac death. Early interference with fibroblast proliferation to limit fibrosis should be able to prevent fibrogenesis from further perpetuating cardiac diseases. However, Ca2+ signaling mechanisms in cardiac fibroblasts (CFs) have remained elusive. Our long-term goal is to investigate the Ca2+ signaling mechanisms in CFs and their potential roles in fibrotic heart diseases. We have previously cloned a novel bi-functional Ca2+-permeable ion channel TRPM7. Our recent preliminary data showed that: 1) TRPM7 is likely the molecular basis of native TRPM7L, the only Ca2+-permeable ion channel identified in the CFs to date; 2) TRPM7 inward currents can be drastically enhanced by a decrease in extracellular pH; 3) oxidative stress significantly increased TRPM7 inward currents; 4) chronic exposure to TGFb1 up-regulates TRPM7 expression in CFs. Given that acidosis, oxidative stress, and TGFb1 are potent stimuli in initiating fibrogenesis during myocardial injury/infarction, we hypothesize that TRPM7L is essential for Ca2+ signaling in CFs, and plays important roles in myocardial ischemia/infarction initiated fibrogenesis. We propose to: 1) determine if TRPM7 is the molecular basis of TRPM7L, and if TRPM7L underlies Ca2+ signaling mechanisms in CFs under oxidative stress and acidosis stimuli; 2) investigate if chronic fibrogenesis stimuli up-regulate TRPM7L; 3) determine if TRPM7L is essential for CFs' functions in responses to fibrogenesis stimuli. Patch-clamp, Ca2+ imaging, biochemical methods and gene knock-down will be used in this project to reveal the potential roles of TRPM7L-mediated Ca2+ signals in fibrogenesis cascade events. The results of the proposed studies will lead to a future in vivo study which is designed to evaluate potential roles of TRPM7 in cardiac fibrosis using TRPM7 knock out mice, and will ultimately provide clinical insights into therapeutic approaches for fibrosis associated cardiac diseases.

Excessive TRPM7 channel activity is linked to neuronal cell death, cancer cell metastasis, and as our preliminary data will demonstrate, the development of cardiac fibrosis in a hypertensive hypertrophy/heart failure mouse model. Collectively, these findings underscore a critical role for TRPM7 in the pathology of a multitude of diseases, making channel an attractive target for therapeutic intervention. However, the specific mechanisms controlling TRPM7 activity in vivo remain unknown. We have made the critical discovery that TRPM7 binds to CNNM proteins (CNNM1-4), which our preliminary data indicate function as regulatory subunits of the channel. We further show that PTP4A phosphatases activate TRPM7 in a CNNM-dependent manner. TRPM7 is the first identified ion channel to possess a kinase domain, the function of which is poorly understood. We recently reported the discovery that auto-phosphorylation of the channel plays a decisive role in controlling the stability of TRPM7 protein expression and the channel's localization in cells. We hypothesize that PTP4A phosphatases, CNNMs, and channel phosphorylation operate in concert to regulate TRPM7. In the multi-PI proposal, we propose three specific aims to elucidate the molecular mechanisms controlling the TRPM7 channel with the long-term goal of understanding how the channel becomes upregulated during cardiac fibrosis. In specific aim 1, we will employ electrophysiology, imaging, and biochemical approaches to elucidate the regulation of TRPM7 by CNNMs and PTP4As. In specific aim 2, we will apply analytical mass spectrometry, biochemical, and imaging approaches to understand how phosphorylation of the channel regulates TRPM7 protein expression and its cellular localization. In specific aim 3, we will investigate the specific mechanism(s) controlling pathological stimulation of the channel during cardiac fibrosis. There is an urgent need for new treatments for stroke, cancer, and heart disease, which kill or severely disable millions of individuals each year. Results from our investigation will have a significant impact by uncovering the mechanisms controlling the channel, which may lead to novel clinical approaches for blocking TRPM7's pathological actions in these devastating diseases.